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J. Agric. Food Chem. 1995,43,2800-2802
ARTICLES Plant Flavonoids, Especially Tea Flavonols, Are Powerful Antioxidants Using an in Vitro Oxidation Model for Heart Disease Joe A. Vinson," Yousef A. Dabbagh, Mamdouh M. Serry, and Jinhee Jang Department of Chemistry, University of Scranton, Scranton, Pennsylvania 18510
Flavonoids, commonly occurring antioxidants in foods, have been compared in a dose-response manner with vitamins C and E and p-carotene and found to be powerful antioxidants using an in uitro lipoprotein oxidation model. This model simulates the oxidation of low-density lipoproteins, which results in atherosclerosis. Of the flavonoids and flavonoid-related compounds, flavonols found in tea are the most powerful natural antioxidants. These results provide a mechanism for the beneficial epidemiological effect of dietary flavonoids on heart disease.
Keywords: Flauonoih antioxidants INTRODUCTION Plants synthesize the well-known antioxidants tocopherols, ascorbic acid, and carotenoids. Flavonoids are phenol derivatives synthesized in substantial amounts (0.5-1.5%) and widely distributed in plants. These compounds have been found to possess antioxidant and free radical scavenging activity in foods (Shahidi and Wanasundara, 1992). More than 4000 individual flavonoids have been identified in higher and lower plants (Harborne, 1988). They are hydroxylated, methoxylated, and/or glycosylated derivatives of the 2-phenylbenzo[alpyrane ring, which consists of two benzene rings combined by mediation of the oxygen-containing pyrane ring. In general, the leaves, flowers, fruit, and other tissues of the plant contain glycosides, woody tissues contain aglycons, and seeds may contain either. They can be subdivided into six classes of flavonoids and flavonoid-related compounds: flavones, flavanones, isoflavones, flavonols, flavanols, and anthocyanins. The basic ring skeletons of these classes are shown in Figure 1. Flavonoids can be considered derivatives of flavonone with the B ring substituted on the 2-position of the heterocyclic ring except for isoflavonoids such as isoflavanone, in which it is substituted on the 3-position. Phenolic acids, while not possessing the ring structure of flavonoids, are phenolic antioxidants in foods and are often esterified with flavonoids. Lower density lipoproteins, in the form of low-density lipoproteins (LDL) and very low density lipoproteins (VLDL), are hypothesized t o be involved in the pathogenesis of atherosclerosis after oxidation (Steinberg et al., 1989). There is substantial epidemiological as well as experimental evidence that increasing amounts of antioxidants in the diet such as vitamins C and E and ,&carotene are preventive for heart disease (Manson et al., 1993). Recently in a Dutch epidemiological study, dietary flavonoid intake, the majority from tea, was found to significantly reduce deaths from heart disease (Hertog et al., 1993). Several flavonoids have been *Author t o whom correspondence should be addressed [telephone (717) 941-7551;fax (717) 941-75101.
0 flavone
0
0 lsoflavone
pB
flavanone
A I
OH
A n B A & : H
OH
0 flavonol
Flavanol
Anthocyanin
Figure 1. Flavonoid subclasses.
found to inhibit LDL oxidation in uitro by cupric ion and by macrophages (DeWhalley et al., 1990; Mangiapane et al., 1992). This research was undertaken to systematically compare flavonoids and other natural antioxidants in a lipoprotein oxidation model that simulates the oxidation mechanism of atherogenesis. METHODS Pure flavonoids and other antioxidantswere obtained from either Sigma Chemical Co. or Aldrich Chemical Co. The tea compounds were a gift from Thomas J. Lipton Tea Co. Cyanidin chloride was synthesized and purified (King and White, 1957). Welch's Grape Color Extract, Type 250, was a gifl of Welch's Co., and silymarin was donated by Madaus AG. Lipoproteins LDL plus VLDL were isolated from the plasma of a single normocholesterolemic individual, who was not
consuming antioxidant supplements, by using an affinity column (Isolabs, Inc.). The procedure for incubation of the oxidant cupric ion with the lipoproteins, analysis of the lipid peroxidation products, and determination of the concentration of antioxidant for 50% inhibition (IC50) has previously been described (Vinson and Hontz, 1995). Phenol concentrationin mixtures was determined with the Folin-Ciocalteu reagent (Sigma)using catechin as the standard. RESULTS AND DISCUSSION All compounds tested had a sigmoidal dose-response inhibition of lipoprotein oxidation. The IC50 data are
0021-8561/95/1443-2800$09.00/00 1995 American Chemical Society
J, Agric. Food Chem., Vol. 43, No. 11, 1995 2801
Plant Flavonoid Antioxidants
Table 1. Comparison of Antioxidant Effectiveness of Vitamins, Flavonoids, and Phenols" comDound OH substitution butylated hydroxyanisole butylated hydroxytoluene trolox ascorbic acid (vitamin C) a-tocophenol (vitamin E) /?-carotene (provitamin A)
Vitamins and Synthetic Phenols mono mono mono vicinal mono none
ICs0 (LAM)
0.181 0.270 1.26 1.45 2.40 4.30
Flavones genistein (isoflavone) apigenin baicalein chrysin flavone
5,7,4' 5,7,4' 5,6,7' 5,7 none
14.3 '16 >16 216 >16
5,7,3' 5,3' 5,3' 5,7,4'
3.66 >16 >16 >16
Flavanones hesperitin hesperidin (hesperetin rutinoside) neohesperidin naringenin Flavonols quercetin taxifolin (dihydroquercetin) myricetin rutin (quercetin rutinoside) morin kaempferol
3,5,7,3',4' 3,5.7,3'.4' 3;5;7;3';4',5' 5,7,3',4' 3,5,7,2',4' 3,5,7,4'
0.224 0.344 0.477 0.512 0.734 1.82
5,7,3',4',5',3",4",5" 3.5.7.3'.4',5' 5;7;3;,4;,3;',4",5" 3,5,7,3',4'
0.075 0.097 0.142 0.187
Flavanols epigallocatechin 3-gallate epigallocatechin epicatechin 3-gallate catechin cyanindin chloride grape skin extract
Anthocyanins 3,5,7,3',4'
0.212 0.951b
Phenolic Acids tannic acid (2-5 gallic esters with glucose) caffeic acid (cinnamic acid) nordihydroguairetic acid (pyrocatechol) chlorogenic acid (cinnamic ester) 3,4-dihydrobenzoic acid gallic acid (benzoic acid) ellagic acid (gallic lactone) p-coumaric acid (cinnamic acid) resveratrol (stilbene) gossypol (dinaphthalene) silymarin (flavolignan) phloretin (propiophenone)
3,4 3,4,3',4' 3,4 3,4 3,4,5 one ortho, one mono 4
0.152 0.241 0.255 0.296 0.455 1.25 2.50 >16
Miscellaneous Phenols 3,5,4' two ortho, two mono 3,5,7, one mono 2,4,6,4'
0.332 0.572 0.853 1.08
a Antioxidants were added in duplicate at various concentrations to 70 pglmL of lipoproteins in a pH 7.4 buffer with 25 pM cupric ion for a period of 6 h at 37 "C. Lipid peroxidation was measured by fluorescence of thiobarbituric acid reaction vs a control with no antioxidant added. The concentration for 50% inhibition was determined graphically. Based on phenol concentration determined according to the Folin method.
shown in Table 1for flavonoids and other natural and synthetic antioxidants. The listing within each group is in the order of decreasing antioxidant potency. In general, the well-known antioxidant vitamins were very poor antioxidants when compared to those flavonoids that had antioxidant activity. Trolox, a water-soluble form of vitamin E, was 2 times more potent than vitamin E, indicating some water solubility is advantageous in this oxidation model. The food additives BHT and BHA were very good antioxidants, probably because they are highly lipophilic and strongly bind t o the lipoproteins. Of the classes of flavonoids, the flavones from petals, leaves, and seeds had the poorest antioxidant activity due t o the lack of o-dihydroxy groups. Genistein, an isoflavone from soybeans, was the only compound to show any activity. The flavanones, primarily found in
citrus fruit, were the next poorest class, with only hesperitin possessing any significant antioxidant character. The flavonols, commonly found flavonoids from flowers, leaves, and fruits, were good antioxidants. In addition to o-dihydroxy groups, they also have an o-hydroxyketo group that can chelate cupric ion (Thompson et al., 19761, rendering it less oxidative. However, chelation is probably not the mechanism of action of flavonoids in our model since the effective antioxidant concentrations are 1-3 orders of magnitude lower than that of the cupric ion in the medium (25 pM). Rutin, the glycoside of quercetin, was a poorer antioxidant than quercetin. This was also found by Afanas'ev et al. (1989). Phenolic acids (hydroxycinnamic,hydroxybenzoic, and ellagitannins) are found in many fruits and vegetables.
Vinson et al.
2802 J. Agric. Food Chem., Vol. 43, No. 11, 1995
Chlorogenic and caffeic acids are present at 250 mg/ cup of coffee (Ohnishi et al., 1994). These compounds, with the exception of p-coumaric acid, which does not possess an o-dihydroxy group, were very good antioxidants. Phenolic acids have been shown by Laranjinha et al. (1994) to be consumed before LDL is oxidized, analogous to vitamin E. Anthocyanins are responsible for the colors of flowers and fruits and are used as colorants for foods and pharmaceuticals as reviewed by Francis (1992). Although only one pure anthocyanin was tested, cyanidin proved t o be a very good antioxidant. The grape skin extract was also a good antioxidant. Acylated malvidin glucoside, a major component of grape skins, was found to have twice the antioxidant power of tocopherol in a linoleic oxidation model (Tamura and Yamagami, 1994). There were also three plant phenols studied that were not flavonoids. Gossypol from cottonseed and silymarin from milk thistle were good antioxidants. Resveratrol was a very good antioxidant and has been touted as an important antioxidant in red wines. Along with flavonoids, it has been hypothesized to be at least partially responsible for the health benefits of red wine (Frankel et al., 1993a). The flavanols, epicatechin polyphenols from tea, were the group of compounds containing the most powerful antioxidants. The activity increased with an increase in the number of o-dihydroxy groups. Epigalloepicatechin gallate, the most potent of all antioxidants tested, has four of these groups. It was 20 times more potent than the best vitamin, ascorbic acid. The present work is the first to systematically compare flavonoids and flavonoid-related compounds t o other phenols in a model that simulates the oxidation process leading to atherosclerosis and heart disease. Flavonoids are potentially beneficial with respect t o the prevention of atherosclerosis, and these results provide a mechanism for the epidemiology study showing the benefits of flavonoids for heart disease. Studies on the antioxidant activity of flavonoids following supplementation are now in progress. LITERATURE CITED Afanas’ev, I. B.; Dorozhko, A. I.; Brodskii, A. V.; Kostyuk, A.; Potapovitch, A. I. Chelating and free radical scavenging mechanism of inhibitory action of rutin and quercetin in lipid peroxidation. Biochem. Pharmacol. 1989,38,1763. De Whalley, C. V.; Rankin, S. M.; Hoult, R. S.; Jessup, W.; Leake, D. S. Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages. Biochem. Pharmacol. 1990,39,1743.
Francis, F. J. A new group of food colorants. Trends Food Sci. Technol. 1992,3,27. Frankel, E. N.; Kanner, J.;German, J. B.; Parks, E.; Kinsella, J. E. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet 1993a,341, 454. Frankel, E.N.; Waterhouse, A. L.; Kinsella, J. E. Inhibition of human LDL oxidation by resveratrol. Lancet 199313,341, 1103. Harborne, J. B. The Flavonoids: Advances in Research since 1980; Chapman and Hall: New York, 1988. Hertog, M. G. L.; Feskens, E. J. M.; Hollman, P. C. H.; Katan, M. B.; Kromhout, D. Dietary antioxidant flavonoids and the risk of coronary heart disease: the Zupthen elderly study. Lancet 1993,342,1007. King, H. G. C.; White, T. Conversion of flavonols into anthocyanidins. J . Chem. SOC. 1957,3901. Laranjinha, J . A. N.; Almeida, L. M.; Madeira, V. M. C. Reactivity of dietary phenolic acids with peroxyl radicals: antioxidant activity upon LDL oxidation. Biochem. Pharmacol. 1994,48,487. Mangiapane, H.; Thomson, J.; Salter, A.; Brown, S.; Bell, G. D.; White, D. A. The inhibition of the oxidation of low density lipoproteins by (+)-catechin, a naturally occurring flavonoid. Biochem. Pharmacol. 1992,43,445. Manson, J. E.; Gaziano, J. M.; Jonsa, M. A.; Hennekens, C. H. Antioxidants and cardiovascular disease: a review. J . Am. Coll. Nutr. 1993,12,426. Ohnishi, M.; Morishita, H.; Iwahashi, H.; Toda, S.; Shirataki, Y.; Kimura, M.; Kido, R. Inhibitory effects of chlorogenic acids on linoleic acid peroxidation and hemolysis. Phytochemistry 1994,36,579. Shahidi, F.; Wanasundara, P. K. Phenolic antioxidants. Crit. Rev. Food Sci. Nutr. 1992,32,67. Steinberg, D.; Parthasarathy, S.; Carew, T. E.; Khoo, J. C.; Witzum, J. L. Beyond cholesterol; modifications of lowdensity lipoproteins. New Engl. J . Med. 1989,320,915. Tamura, H.; Yamagami, A. Antioxidant activity of monoacylated anthocyanins isolated from Muscat Bailey A grape. J . Agric. Food Chem. 1994,42,1612. Thompson, M.; Williams, C. R.; Elliot, G. E. P. Stability of flavonoid complexes of copper (11)and flavonoid antioxidant activity. Anal. Chim. Acta 1976,85, 375. Vinson, J. A.; Hontz, B. A. Phenol antioxidant index: comparative antioxidant effectiveness of red and white wines. J . Agric. Food Chem. 1995,43,401. Received for review July 19, 1995. Accepted September 21, 1995.@This work was supported in part by the Thomas J. Lipton Tea Co. and by a generous gift of columns from Michael Jackson of Isolab, Inc.
JF9504982
Abstract published in Advance ACS Abstracts, November 1, 1995. @
